ABSTRACT Young skeletal muscle displays remarkable resilience following an acute injury event, evidenced by a robust regenerative response and functional recovery. In contrast, even a relatively minor injury to aged muscle can result in significant functional impairments owing to compromised regeneration. Numerous studies have identified muscle stem cells (MuSCs) as a major culprit in the failed healing response of aged muscle. MuSCs represent a reserve cell population that play a primary role in muscle regeneration. However, with aging, MuSCs display a myogenic-to-fibrogenic conversion, resulting in fibrosis at the expense of myofiber regeneration. Although cell-autonomous deficits play an important role in cellular declines with aging, the contribution of biophysical cues from the surrounding microenvironment has been increasingly appreciated. Tissue regeneration involves a tightly-regulated and bi-directional communication between stem cells and their biophysical microenvironment. Elegant in vitro studies have demonstrated that substrates engineered to mimic the elasticity typical of young, healthy muscle promoted stem cell myogenicity, whereas stiffer substrates drove stem cell chondrogenic/osteogenic differentiation. In vivo, compositional and physical changes in the extracellular matrix (ECM) similarly exert deleterious effects on stem cell function. We and others have shown that age-related alterations in ECM biophysical features contribute to disrupted MuSC lineage specification. Whereas the bulk of studies to date, including our own, have focused on the direct effects of the ECM on stem cell responses through mechanotransductive signaling cascades, our latest data suggest a novel role of extracellular vesicles (EVs) in mediating the effect of the ECM on stem cell responses. We have found that substrates engineered to mimic the stiffness of aged skeletal muscle promoted the cellular release of EVs that inhibited MuSC myogenicity. In contrast, EVs released by cells seeded on substrates designed to mimic the stiffness of young skeletal muscle induced robust myotube formation. Aim 1 studies will investigate whether and how aging of the skeletal muscle ECM shifts the molecular cargoes of muscle EVs, resulting in compromised MuSC myogenicity and functional regeneration. While aging is associated with ECM stiffening, mechanical loading increases tissue elasticity and promotes ECM remodeling. Exercise has also been associated with enhanced muscle regeneration, though many questions remain regarding the mechanisms underlying these benefits. Our data suggest that muscle activity shifts EV cargoes in favor of enhanced MuSC myogenic lineage specification, effects we hypothesize to be responsive to ECM remodeling. Aim 2 studies will mechanistically interrogate the ability of muscle activity to rejuvenate ECM biophysical features and the biomolecular signatures of EVs in support of MuSC myogenicity and functional muscle regeneration.